Respiratory epithelium is critical in protecting humans from disease and acts as a barrier to invading microbes present in the air. Airway epithelial cells defend the host physiology by blocking paracellular permeability, modulating airway function through cellular interactions, and transporting inhaled microorganisms away via ciliated epithelial cells (Bals and Hiemstra, 2004, Cotran et al, 1999). Epithelial cells are regulators of the innate immune response and also induce potent immunomodulatory and inflammatory mediators (cytokines and chemokines), thus recruiting phagocytic and inflammatory cells and facilitating microbial destruction (Bals and Hiemstra, 2004; Knight and Holgate, 2003).
The respiratory epithelia defend the host through a complex multi-layered system of pseudo-stratified epithelial cells, a basement membrane, and underlying mesenchymal cells (Hiemstra and Bals, 2004). Ciliated, secretory, and basal epithelial cells are joined by intercellular junctions and anchored to the basement membrane via desmosomal interactions. Through tight junctions and the mucociliary layer, the basement membrane maintains polarity of the epithelium and presents a physical barrier between the mesenchymal layer and the airway (Knight and Holgate, 2003; Gibson and Perrimon, 2003). Spatial cellular relationships, cell membrane junctions, extracellular matrices (e.g., basement membrane and ground substances), and soluble signals (endocrine, autocrine, and paracrine) influence tissue differentiation. Complex recapitulated 3D models must emulate these complex cellular relationships to model characteristics of in situ airway epithelium.
Current models of in vivo lung epithelium are limited by fidelity of the model and scale. Traditional two-dimensional (2D) monolayer cultures such as immortalized human epithelial cell lines and primary normal human bronchial epithelial (NHBE) cells as well as air-liquid interface cultures (3D) fail to express the innate tissue fidelity characteristic of normal human respiratory epithelia (Carterson et al., 2005). Thus, their state of differentiation and intracellular signaling pathways differ from epithelial cells in vivo. Recently, 3D aggregates derived from an alveolar epithelial tumor cell line (A549) were used as targets for bacterial infection (Carterson et al., 2005). While superior to two dimensional cultures, the 3D aggregates lacked the functional and structural characteristics of airway epithelium in situ. Primary isolates of HBE cells provide a pseudo-differentiated model with structure and function similar to epithelial cells in vivo; however, this fidelity is short-lived in vitro (Gray et al, 1996). Air-liquid interface cultures of primary HBE cells (or submerged cultures of human adenoid epithelial cells Wright et al, 2005) are grown on collagen-coated filters in wells, on top of a permeable filter. These cells receive nutrients basolaterally and their apical side is exposed to humidified air. The result is a culture of well-differentiated heterogeneous (ciliated, secretory, basal) epithelial cells essentially identical to airway epithelium in situ (Adler and Li, 2001). Although this model mimics the fidelity of the human respiratory epithelium in structure and function, maintenance of consistent cultures is difficult, time consuming, and restricted to small-scale production.
Culturing normal 3D epithelium configurations larger than 3 mm is problematic using traditional in vitro culture technology. Short-term cultures have been accomplished but, long-term growth requires sophisticated, defined culture media or in vitro transformation to increase longevity. To address this, horizontally rotating cylindrical tissue culture vessels or rotating wall vessels (RWV) developed at NASA's Johnson Space Center (Schwarz et al, U.S. Pat. No. 5,026,650) have been used to model many 3D tissues (Goodwin et al, 1988, 1992, and 1993) (Table 1). This technology allows the recapitulated tissues to be used as host targets for viral infectivity (Goodwin et al., 2000) by providing controlled supplies of oxygen and nutrients, with minimal turbulence and extremely low shear (Schwarz et al, 1992). These vessels rotate the wall and culture media inside at identical angular velocity, thus continuously randomizing the gravity vector and holding particles such as microcarriers and cells relatively motionless in a quiescent fluid (Schwarz et al 1992; Tsao et al, 1992).
TABLE 13D TISSUES ENGINEERED IN THE ROTATING WALL VESSELNORMALRefBovine Cartilage (chondrocytes)(Baker, 1997)Rat Cardiomyocytes(Bursac, 2003)Human Bone (Osteoblast)(Klement, 2004; Wang, 2005)Human Cornea(O'Connor, 1999)Human Kidney(Goodwin, 1993; Hammond, 1997)Human Liver(Yoffe, 1999)Human Lymphoid(Margolis, 1997; Pellis, 1997)Human Neural Progenitor(Goodwin, 2003; Goodwin, 2005)Human Renal Proximal Tubule(Hammond, 1997)Human Small Intestinal Epithelial(Goodwin, 1993)CANCERHuman Colon(Goodwin, 1988; Goodwin, 1992)Human Lung(Vertrees, 2005)Human Ovarian(Goodwin, 1997)Human Prostate(Wang, 2005)
Optimally, a cell-based respiratory epithelia model would reproduce the structural organization, multicellular complexity, differentiation state, and function of the human respiratory epithelium. Here we report the successful engineering of the first in vitro model of the human respiratory epithelium using primary mesenchymal hBTCs as the foundation matrix and an adult HBE immortalized cell line BEAS-2B as the overlying component. The RWV culture system provides ease of manipulation, consistency in culture conditions, and well-differentiated TLAs that share structural and functional characteristics of the human respiratory epithelium. When combined with a solid matrix, cocultivation of epithelial and mesenchymal cells in RWVs allow cells to auto assemble into 3D tissue-like masses that we postulate fulfill four of the five basic stages of tissue regeneration and differentiation (FIG. 2). Like the air-liquid interface model (O'Brien et al, 2002), the epithelial cell organization of the TLAs improves the expression of airway epithelial characteristics, and also cellular communication. Thus, TLAs represent a physiologically relevant model of the human respiratory epithelia that can be used in large-scale production for prolonged periods.
TABLE 2ABBREVIATIONSAbbrTerm2DTwo-Dimensional3DThree-DimensionalATCC ®American Tissue-type Culture CollectionBEBroncho-EpithelialBMEEagle's Basal MediumBSABovine Serum AlbuminBTCBroncho-Tracheal CellsBVBudding VirusCFCystic FibrosisCMF-PBScalcium- and magnesium-free PBSDEAEDiethylamino EthanolDMEMDulbecco's MEMDPBSDulbecco's PBSECMExtracellular MatrixEMAEpithelial Membrane AntigenEMEMEagle's MEMFBSFetal Bovine SerumFVIIIFactor VIIIGTSFGlucose Trisugar FormulaH&EHaematoxylin and EosinhBEHuman Broncho-eptihelialhBTCHuman Mesenchymal BTCHIVHuman Immunodeficiency VirusICAMIntercellular Adhesion MoleculeIHCImmunocytochemistryIMDMIscove's Modified Dulbecco's MediumMEMMinimal Essential MediumMOIMultiplicity of InfectionMVMicrovilliNHBEPrimary Normal hBEPBSPhosphate-Buffered SalinePECAMPlatelet/Endothelial Cell Adhesion MoleculepfuParticle Forming UnitspiPost InfectionPIVParainfluenza VirusRARβRetinoic Acid Receptor betaRSVRespiratory Syncytial VirusRWVRotating Wall VesselSEMScanning Electron MicrographSPASurfactant Protein ASPGSucrose-Phosphate-Glyoxylic acidTEMTransmission Electron MicrographTJTight JunctionTLATissue-Like Assemblies (3D-HBE)VVacuoleVNCVirus NucleocapsidwtWild-TypeZOZonula Occludens